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How dry are anhydrous enzymes? Measurement of residual and buried 18 O‐labeled water molecules using mass spectrometry
Author(s) -
Dolman Mark,
Halling Peter J.,
Moore Barry D.,
Waldron Susan
Publication year - 1997
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(199703)41:3<313::aid-bip6>3.0.co;2-v
Subject(s) - chemistry , anhydrous , bound water , molecule , mass spectrometry , lysozyme , water of crystallization , chromatography , analytical chemistry (journal) , organic chemistry , biochemistry
There is continual debate over the central role of water in protein folding, structure, stability, and dynamics. Catalytic activity has been demonstrated in organic media with, apparently “anhydrous” enzymes. Hence there is considerable discussion over whether there are a few residual water molecules or if the enzymes are demonstrating activity in the complete absence of water. Here we present measurements designed to test this hypothesis based on the detection of 18 O‐labeled water by mass spectrometry. This extremely sensitive technique avoids many of the potential errors associated with published methods for measuring water content such as gravimetry or Karl Fischer titrations. We have also explored the mass spectrometric detection of 2 H‐enriched water and found that lyophilization of deuteron‐labeled protein can lead to extensive loss of the isotopic label during the drying process. “Anhydrous” protein was produced by extended drying, over P 2 O 5 , of lyophilized powders hydrated through the vapor phase with 18 O‐labeled water. Redissolution in standard water released the remaining protein‐bound 18 O‐labeled water molecules, and the isotopic enrichment of the water was used to calculate the number of bound molecules per mole of protein. In the cases of lysozyme and subtilisin Carlsberg, 4 ± 2 and 15 ± 2 waters per mole were found, respectively. Comparisons with crystal structures showed these values correspond closely to the expected number of buried water molecules in these proteins. This is consistent with the idea that water physically entrapped within the rigid protein structure is retained but all the other more accessible surface‐bound hydration molecules can be removed by the drying process. Such anhydrous subtilisin Carlsberg preparations have been found to be weakly catalytic and therefore it appears that additional water molecules on the surface of the enzyme are not essential for this level of enzyme activity. © 1997 John Wiley & Sons, Inc.